Compositions and methods for detecting s-nitrosylation and s-sulfinylation

ABSTRACT

The present invention relates to methods for detecting protein S-sulfinylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to pending U.S. Provisional Patent Application No. 61/820,401, filed May 7, 2013, the contents of which are incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for detecting protein S-nitrosylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.

BACKGROUND OF THE INVENTION

Reversible redox post-translational modifications on protein thiols have been implicated in several signaling pathways of biological importance. Protein S-nitrosylation and proteins S-sulfinylation are two of these modifications that play critical roles in maintaining the redox balance of proteins. Redox imbalance has recently been shown to play a crucial role in heart disease, neurodegeneration and cancer. Protein S-nitrosylation describes the reversible, post-translational modification of select thiols with nitric oxide (NO) and/or its oxidized products to form S-nitrosothiols (SNO). Protein S-sulfinylation describes the oxidation of cysteine thiols to a SOOH (sulfinic acid) motif via a peroxide-mediated pathway.

Current methods to study S-nitrosylation and S-sulfinylation lack sufficient selectivity, and may over-represent the functional role of S-nitrosylation and S-sulfinylation.

For example, several methods have been reported to label and enrich sites of protein S-nitrosylation, including several versions of the biotin switch technique (BST), gold nanoparticle based enrichment, organomercury based methods and phosphine-based probes. By far, the most popular method is BST, which relies on ascorbate reduction to selectively reduce sites of S-nitrosylation. Sodium ascorbate has been shown to reduce activated disulfides and its reactivity with cysteine sulfenic acids and thiocysteines has not been thoroughly investigated. This suggests that the BST may be contaminated with weak disulfides, thiosulfhydrylation (R-SSH), or other activated thiol modifications. Such findings demonstrate that knowledge about SNO modifications derived from such methods are indirect.

New selective methods for detecting S-nitrosylation and S-sulfinylation are needed.

SUMMARY OF THE INVENTION

The sulfhydryl group of the amino acid cysteine is a key target of oxidative stress, and is readily modified to induce temporary and sometimes permanent protein damage (see, e.g., Paulsen, C. E. & Carroll, K. S. Chem Rev 113, 4633-79 (2013)). Depending on the abundance and type of radical species, cysteine is covalently modified to one of a series of distinct chemical moieties. For example, reactive nitrogen species can induce the formation of S-nitrosocysteine (R-SNO) (see, e.g., Majmudar, J. D. & Martin, B. R. Biopolymers (2013)), while reactive oxygen species can induce formation of S-sulfenylcysteine (R-SOH) (see, e.g., Paulsen C. E. & Carroll, K. S. Chem Rev 113, 4633-79 (2013)). These modifications are unstable and transient, and often act to temporarily inactivate functional cysteines (see, e.g., Nakamura, T. et al. Neuron 78, 596-614 (2013)). In some cases, reversible cysteine modifications have evolved as redox regulatory switches that transmit or enhance cellular cues (see, e.g., Paulsen, C. E. & Carroll, K. S. Chem Rev 113, 4633-79 (2013)). When these mechanisms are left unchecked, irreversible oxidation can occur (such as R—SO₂H, R—SO₃H), decorating the proteome with oxidative damage.

Nitric oxide is a critical second messenger involved in vasorelaxation, immunity, and neurotransmission. Under oxidative conditions, elevated nitric oxide promotes covalent S-nitrosylation of cysteine thiols in proteins. Aberrant S-nitrosylation is implicated in the pathology of many diseases, including inflammation, stroke, and neurodegeneration. Both the ascorbate-dependent biotin-switch (see, e.g., Jaffrey, S. R. & Snyder, S. H. Sci STKE 2001, pl1 (2001)) and organomercury enrichment (see, e.g., Doulias, P. T. et al. Sci Signal 6, rs1 (2013)) have been used to annotate protein S-nitrosylation by mass spectrometry. Using these methods, hundreds of endogenous S-nitrosylated proteins have been identified, highlighting particular enrichment of abundant metabolic enzymes with nucleophilic or redox-active thiols (see, e.g., Doulias, P. T. et al. Sci Signal 6, rs1 (2013)). While both methods have led to important biological revelations of the significance of S-nitrosylation, more robust methods are necessary (see, e.g., Forrester, M. T., Foster, M. W. & Stamler, J. S. J Biol Chem 282, 13977-83 (2007); Giustarini, D. et al. Nitric Oxide-Biology and Chemistry 19, 252-258 (2008)). The ascorbate-dependent biotin-switch can lead to reduction of weak disulfides (see, e.g., Giustarini, D. et al. Nitric Oxide-Biology and Chemistry 19, 252-258 (2008)) and other reversible cysteine modifications (see, e.g., Reisz, J. A. et al. FEBS J 280, 6150-61 (2013)), which can scramble with existing disulfides. Similarly, the selectivity of organomercury enrichment has not been thoroughly explored, and may introduce false-positives after performic acid oxidation of disulfide-capped thiols, and oxidize other amino acid side chains. While emerging triarylphosphine-catalyzed methods for detecting nitrosothiol are promising (see, e.g., Seneviratne, U. et al. J Am Chem Soc 135, 7693-704 (2013)), these methods have not been thoroughly validated in complex proteomes.

Experiments conducted during the course of developing embodiments for the present invention showed sulfinic acids and nitrosothiols to react to form a thiosulfonate bond. This reactivity was leveraged to enrich and annotate ˜1000 endogenous S-nitrosylated proteins (see, FIG. 16). In parallel, S-nitrosothiol probes were used to label endogenous S-sulfinylated proteins, demonstrating a direct, bi-directional method to profile distinct cysteine modifications based on the inherent reactivity of sulfinic acids. As such, the present invention provides new chemoselective ligation methods for the detection of protein S-nitrosylation and S-sulfinylation that simplifies and improves the detection of these redox modifications.

Accordingly, in certain embodiments, the present invention provides methods for detecting protein S-nitrosylation within thiol groups in proteins, metabolites, or materials. In particular, the present invention provides methods for detecting protein S-nitrosylation within cysteine residues of a protein, comprising providing a biological sample comprising proteins having cysteine residues and a composition comprising a labeled sulfinic acid moiety (e.g., a sulfinic acid probe), exposing the composition to the biological sample such that the labeled sulfinic acid moiety is able to interact with the nitrosothiols on cysteine side chains of the protein so as to generate labeled sulfonothioate moieties, and characterizing the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-nitrosylation. In some embodiments, the methods further comprise identifying the protein having been characterized as having undergone S-nitrosylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-nitrosylation.

In some embodiments, the labeled sulfinic acid moiety comprises a labeling agent (e.g., a fluorescent dye detectable by in-gel fluorescence) and/or an enrichment agent (such as biotin and/or desthiobiotin for affinity enrichment). In some embodiments, flash chromatography is used to characterize the cysteine residues of a protein having sulfonothioate moieties as having undergone S-nitrosylation. In certain embodiments, flash chromatography (normal or reverse phase) is used to purify the sulfonothioate adduct following reaction between a nitrosothiol and a sulfinic acid to validate probe reactivity. In some embodiments, high performance liquid chromatography is used to characterize the cysteine residues of a protein having sulfonothioate moieties as having undergone S-nitrosylation by reaction with a labeled sulfinic acid moiety. In some embodiments, mass spectrometry is used to characterize the cysteine residues of a protein having sulfonothioate moieties as having undergone S-nitrosylation by reaction with a labeled sulfinic acid moiety.

In some embodiments, the biological sample is an in vivo sample, an ex vivo sample, or an in vitro sample. In some embodiments, the biological sample is a mammalian biological sample. In some embodiments, the biological sample is a human biological sample.

In certain embodiments, the present invention provides methods for identifying pharmaceutical agents capable of inhibiting S-nitrosylation, comprising providing a pharmaceutical agent and a biological sample comprising proteins having cysteine residues known to undergo S-nitrosylation, exposing the pharmaceutical agent to the biological sample, characterizing the cysteine residues of a protein having S-nitrosothiol moieties as having or not having undergone S-nitrosylation with the methods for detecting protein S-nitrosylation within cysteine residues of a protein (described herein), and identifying the pharmaceutical agent as a S-nitrosylation inhibitor if the characterization indicates an absence of S-nitrosylation.

In certain embodiments, the present invention provides methods for detecting protein S-sulfinylation within cysteine residues of a protein, comprising providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled nitrosothiol moiety, exposing the composition to the biological sample such that the labeled nitrosothiol moiety is able to interact with sulfinic acid side chains of the cysteine residues so as to generate labeled sulfonothioate moieties, characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation.

In some embodiments, the methods further comprise identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation.

In some embodiments, the labeled nitrosothiol moiety is a labeled nitrosoglutathione moiety. In some embodiments, the labeled nitrosothiol moiety is a labeled S-Nitroso-N-acetylpenicillamine moiety.

In some embodiments, labeled nitrosothiol moiety comprises an imaging agent and/or an enrichment tag. In some embodiments, the imaging agent is a fluorescent dye. In some embodiments, the in-gel fluorescence is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation. In some embodiments, the enrichment agent is biotin or desthiobiotin.

In some embodiments, flash chromatography followed by NMR and/or mass spectrometry is used to structurally identify the product of the reaction between a sulfinic acid side chain of a cysteine residue and a labeled nitrosothiol moiety.

In some embodiments, high performance liquid chromatography is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation.

In some embodiments, mass spectrometry is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties moieties as having undergone S-nitrosylation.

In some embodiments, the biological sample is an in vivo sample, an ex vivo sample, or an in vitro sample.

In some embodiments, the biological sample is a mammalian biological sample. In some embodiments, the biological sample is a human biological sample.

In certain embodiments, the present invention provides methods for identifying pharmaceutical agents capable of inhibiting S-sulfinylation, comprising providing a pharmaceutical agent and a biological sample comprising one or more proteins having cysteine residues known to undergo S-sulfinylation, exposing the pharmaceutical agent to the biological sample, characterizing the cysteine residues of a protein having labeled sulfonothioate moieties as having or not having undergone S-sulfinylation with the methods for detecting protein S-sulfinylation within cysteine residues of a protein, and identifying the pharmaceutical agent as a S-sulfinylation inhibitor if the characterization indicates an absence of S-sulfinylation.

In certain embodiments, the present invention provides methods for annotating and profiling the exact amino acid sites on proteins that have undergone S-nitrosylation or S-sulfinylation. For example, in some embodiments such methods utilize “in-built” cleavable linkers (such as, for example, dialkyl or diaryl dialkoxysilanes, orthoesters, vinyl ethers and such) to enable cleavage of a sulfonothiaote adduct (post reaction with a nitrosothiols) thereby leaving behind a chemical reporter tag that can be utilized as a mass-spectrometric signature for the particular amino acid. In some embodiments, the sulfonothioate adduct may itself be used as a cleavable linker (e.g., cleaved with tris-carboxy-ethyl phosphine or similar reducing agents) in presence of an orthogonal thiol capture reagent to provide a mass spectrometry reporter ion.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Sulfinic acid probes detect endogenous S-nitrosylation. (a) Phenylsulfinic acid reacts with N-acety-S-nitroso-cysteine methyl ester to form a thiosulfonate bond. (b) Biotin-hypotaurine (biotin-SO₂H) reacts with S-nitrosylated proteins to form a thiosulfonate linkage. Lysates are denatured and reduced thiols are alkylated with iodoacetamide to prevent disulfide exchange. (c) Biotin-SO₂H labels distinct mammalian proteins, but biotin-taurine (biotin-SO₃H) is unreactive. (d) Biotin-SO₂H labeling is enhanced by the nitric oxide donor MAHMA-NONOate. (e) Biotin-SO₂H labeling is unaffected by the sulfenic acid probe dimedone.

FIG. 2. Sulfinic acids react with S-nitrosothiols to form thiosulfonates. A 5 mM solution of N-Acetyl-(S-nitroso)-cysteine methyl ester in phosphate buffered saline (PBS, pH 7.4) was treated (in the dark) with a 20 mM aqueous solution of phenylsulfinic acid in PBS. After 60 minutes, the reaction mixture was separated using gradient elution (5% ACN to 95% ACN over 15 minutes) using an Atlantis C₁₈ reverse phase column, and the absorbance was measured at 281 nm. The two reactants were also individually analyzed under identical conditions (HPLC traces in green and blue). The HPLC trace unambiguously shows the reactivity of sulfinic acids and nitrosothiols to form thiosulfonates (retention time 11.1 min). The thiosulfonate product was separately purified and characterized (HPLC, NMR, MS analysis). Similar results were obtained with hypotaurine (an aliphatic sulfinic acid). However, hypotaurine does not absorb in the UV-vis spectrum, so phenylsulfinic acid was throughout our studies as a model sulfinic acid for HPLC assays.

FIG. 3. Sulfinic acids do not react with iodoacetamide. A 10 mM solution of phenylsulfinic acid was allowed to react with a 20 mM solution of iodoacetamide in potassium phosphate buffer pH 7.4 for 5 hours. After 5 hours, the reaction mixture was injected on an HPLC and separated using gradient elution (5% ACN to 95% ACN over 15 minutes). Absence of additional peaks (over 202-798 nm, monitored on a PDA detector) was interpreted as no reaction between the sulfinic acid and iodoacetamide.

FIG. 4. Sulfinic acids do not react with thiols, disulfides, or aldehydes. A 2 mM solution of phenylsulfinic acid was allowed to stand with 20 mM solutions of N-acetyl-cysteine-methyl ester, benzaldehyde, cystine dimethyl ester, and 5,5′-dithiobis-(2-nitrobenzoic acid) in phosphate buffer pH 7.4 for 5 hours. The reaction mixtures were injected on an HPLC and analyzed using gradient elution (5% ACN to 95% ACN over 15 minutes). Peaks beyond minute 8.6 in (a) are contaminants present in the commercial cysteine sample (Sigma-Aldrich). The absence of any reaction with benzaldehyde suggests no reaction occurs with carbonylated amino acids.

FIG. 5. The thiosulfonate product is reduced by TCEP, but not ascorbate. A 0.5 mM solution of the thiosulfonate product was incubated with 2.5 mM TCEP or 2.5 mM sodium ascorbate in in phosphate buffer (pH 7.4) in the dark at room temperature. After 5 minutes and 60 minutes, an aliquot injected for HPLC analysis and separated using gradient elution (5% ACN to 95% ACN over 15 minutes). TCEP immediately cleaves the thiosulfonate bond, while ascorbate does not after 60 minutes of co-incubation.

FIG. 6. GSNO is consumed by increasing concentrations of sodium phenylsulfinate. (a) A 2 mM solution of S-nitrosoglutathione (GSNO, Cayman) was treated with increasing concentrations of phenylsulfinic acid, and the absorbance of GSNO was monitored over a period of 90 minutes at 340 nm using a plate reader in varyng pH buffers. (b) Calculation of rate constants at differing pH values. At pH 7, the rate becomes more hyperpbolic.

FIG. 7. Determination of reaction efficiency and quantification of products and by-products. (a) Reaction between GSNO and 4-Me-phenylsulfinic acid. The products 19 and 20 were quantified by LC-MS. The product of GSNO and phenylsulfinic acid was not resolved from products during HPLC purification, and was excluded from further analysis. (b) LC-MS standard curves were generated from the HPLC purified thiosulfonate product (19) and commercial 4-Me-piloty's acid (20). (c) Quantification of thiosulfonate product and the 4-Me-piloty's acid side product by LC-MS, presented as 3 replicates with standard deviations.

FIG. 8. Dose-dependent labeling of S-nitrosylated proteins by biotin-SO₂H. 293T and PANC1 cells were lysed in 6 M urea/PBS and alkylated with 50 mM iodoacetamide for 30 minutes, followed by incubation with increasing concentrations of biotin-SO₂H for 45 minutes.

FIG. 9. Hypotaurine competes with biotin-SO₂H. 293T cell lysates were pre-incubated with 5 mM or 50 mM hypotaurine for 30 minutes, followed by treatment with 50 mM iodoacetamide for 30 minutes. The lysate was then labeled with biotin-SO₂H for 30 minutes, separated by SDS-PAGE, and transferred to nitrocellulose for streptavidin detection of endogenous S-nitrosylation.

FIG. 10. Dose-dependent enhancement of S-nitrosylation by the nitric oxide donor (MAHMA NONOate). 293T cell lysates were treated with increasing concentrations of nitric oxide donor, MAHMA NONOate for 5 minutes, followed by treatment with 50 mM iodoacetamide for 20 minutes to alkylate free thiols. S-nitrosylated proteins were detected by incubation with fluorescein-SO₂H for 45 minutes, and separated by SDS-PAGE for in-gel fluorescence detection.

FIG. 11. S-nitrosylation is reduced by ascorbate, and thiosulfonates are reduced by TCEP. 293T cell lysates were treated with 50 mM iodoacetamide for 30 minutes to alkylate any reduced thiols. Next, sodium ascorbate was added for 30 minutes to reduce S-nitrosothiols. Lysates were then treated with 400 μM biotin-SO₂H for 30 minutes, and analyzed by non-reducing SDS-PAGE and streptavidin blotting. Ascorbate efficiently blocks biotin-SO₂H detection of endogenous S-nitrosylation. Next, lysates labeled with biotin-SO₂H were treated with 1 mM TCEP for 15 minutes, demonstrating complete loss of labeling by reduction of the thiosulfonate linkage and confirms there are no TCEP-resistant covalent adducts formed.

FIG. 12. Sulfenic acids are eliminated by ascorbate reduction or denaturing buffers. Lysates were labeled for 1 hour with 1 mM dimedone-alkyne, and precipitated with chloroform:methanol. The protein fraction was sonicated in PBS and diluted to 1 mg/mL, and incubated with 20 μM TAMRA-azide, 1 mM CuSO₄, and 100 μM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) for 1 hour, boiled in reducing loading buffer, and separated by SDS-PAGE. After transfer and Cy5-streptavidin binding, the gel was visualized using a GE Typhoon fluorescence scanner. Dimedone alkyne (compound 14) detection of sulfenic acids in 293T cell lysates is most efficient in PBS. When the lysate is denatured in 6 M urea/PBS, dimedone labeling is nearly completely eliminated. Pre-treatment with sodium ascorbate (20 mM) for 30 minutes efficiently reduces sulfenic acids. These data demonstrates sulfenic acids are eliminated under the denaturing conditions used for biotin-SO₂H labeling of endogenous S-nitrosylated proteins. Furthermore, ascorbate reduces sulfenic acids, and confirms non-selective enrichment of an additional cysteine oxidative modification by the biotin-switch method.

FIG. 13. Iodoacetamide alkylates all free thiols. 293T cell lysates were treated with commercially available iodoacetamide-TAMRA (100 μM, lane 1), or with 50 mM iodoacetamide (30 minutes pre-incubation, room temperature) followed by iodoacetamide-TAMRA (100 μM, lane 2). 50 mM iodoacetamide blocks all labeling by iodoacetamide-TAMRA, providing evidence that biotin-GSNO labels sulfenic acids, and not does not lead to disulfide formation with un-blocked thiols.

FIG. 14. Profiling fluorescein-SO₂H reactivity and selectivity on S-nitrosylated GAPDH. Recombinant human GAPDH was resuspended in 6 M urea/PBS, treated with 50 mM iodoacetamide for 30 minutes, and mixed with dimedone, ascorbate, peroxide or NO donor (5 minutes). Fluorescein-SO₂H (500 μM) was then added to the GAPDH sample for 45 minutes. S-nitrosylated human GAPDH labeling increases upon incubation with the NO donor MAHMA-NONOate, and decreases following treatment with ascorbate. The control probe, fluorescein-SO₃H, does not label GAPDH. Fluorescein-SO₂H labeling is unaffected by pre-incubation with peroxide or dimedone.

FIG. 15. Biotin-SO₂H reacts with human GAPDH and forms a thiosulfonate. Recombinant human GADPH was labeled with biotin-SO₂H for site-specific analysis of S-nitrosylation by high-resolution mass spectrometry.

FIG. 16. Quantitative analysis of biotin-SO2H enrichment in 293T cell lysates. Samples were prepared in two sets, one with biotin-SO2H in the “Light” matched with biotin-SO3H in the “Heavy” (N=2), and the other in reverse (N=2). Each was ran as two technical replicated and SILAC ratios (biotin-SO2H/biotin-SO3H) were combined across experiments. “Directions Detected” describes the set of SILAC samples where the protein was quantified. FORWARD refers to biotin-SO2H (light)/biotin-SO3H (heavy), and REVERSE is the opposite direction, biotin-SO2H (heavy)/biotin-SO3H (light). A ratio of 1000 was assigned to proteins with an infinite ratio, meaning there was not detectable signal in the SILAC pair labeled with biotin-SO3H. Label-free quantification was performed using the Top3 method from the biotin-SO2H labeled pair.

FIG. 17 Annotation and label-free quantification of proteins from 293T whole cell lysates.

FIG. 18. S-nitrosylated protein classification and stoichiometry. (a) Gene ontology (GO) term classification of biotin-SO₂H enriched, S-nitrosylated proteins and unenriched proteins identified by LC-MS analysis. GO term results common across separate categories are denoted as “shared”, and those solely classified in one category are denoted as “unique”. Structural, nucleotide binders, and catalytic activity are displayed on an expanded scale (right). (b) Histogram of S-nitrosylated proteins compared to their corresponding enrichment efficiency. Higher ratios signify more efficient enrichment (and higher stoichiometry) compared to total protein abundance.

FIG. 19. Ratio of S-nitrosylation enrichment to total abundance. Label-free abundance was derived from Top3 quantification.

FIG. 20. S-nitrosothiol probes detect endogenous S-sulfinylation. (a) Direct conjugation of human DJ-1 (Cys106-SO₂H) with N-acetyl-S-nitrosocysteine methyl ester. (b) MS/MS assignment of product ions from the S-sulfinylated peptide of human DJ-1, confirming direct thiosulfonate formation. (c) Gel-based analysis of S-sulfinylation in mammalian lysates with biotin-GSNO. Lysates are denatured and pre-alkylated with iodoacetarnide.

FIG. 21. S-nitroso-N-Acetyl-Cysteine methyl ester reacts with oxidized DJ-1 to form a thiosulfonate. Data was collected in MS^(E) mode.

FIG. 22. HPLC trace of N-acetyl-cysteine-OMe starting material and the S-nitroso product formed upon reaction with sodium nitrite in 1 N HCl and methanol.

FIG. 23. Spectra of (a) ¹H NMR of compound 3. (b) ¹³C NMR of compound 3.

FIG. 24. Spectra of (a) Proton NMR of compound 8 (b) Carbon NMR of compound 8.

FIG. 25. HPLC trace of reaction mixture of biotin-GSNO, compound 16; Peak at minute 13.4 represents product as confirmed by MS analysis.

FIG. 26 shows (A) reaction of sulfinic acids with S-Nitrosocysteine. (B) HPLC traces of starting materials and product in a standard gradient. Traces shown for purified and isolated product.

FIG. 27 shows sulfinic acid probe labels nitrosylated proteins in cell lysates. Labeling was eliminated by ascorbate pretreatment. Gel analyzed using a GE Typhoon laser fluorescence scanner (ex. 488 nm, em. 535/25). No reducing agent added to loading buffer.

FIG. 28 shows the reaction between maleimide and a sulfinic acid.

DETAILED DESCRIPTION OF THE INVENTION

S-nitrosylation involves the covalent incorporation of a nitric oxide moiety into thiol groups, to form S-nitrosothiol (SNO). Where the thiol group belongs to a subset of specific cysteine residues in proteins, the resulting SNO is an S-nitrosoprotein. S-Nitrosylation is a form of post-translational protein modification with similarities to phosphorylation (see, e.g., Anand P, Stamler J S., J. Mol. Med. 90(3): 233-244 (2012)). Generally, S-Nitrosylation operates as a signaling mechanism in that it is stimulus evoked (see, e.g., Hoffmann, J, Dimmeler, S, Haendeler, J. FEBS Lett. 551:153-158 (2003)), precisely targeted (see, e.g., Sun J H, Xin C L, Eu J P, Stamler J S, Meissner G. Proc. Natl. Acad. Sci. U S A 98:11158-11162 (2003), reversible (see, e.g., Padgett C M, Whorton A R. Am. J. Physiol. 269:739-749 (1995)), spatiotemporally restricted (see, e.g., Fang M, Jaffrey S R, Sawa A, Ye K, Luo X, Snyder S H. Neuron 28:183-193 (2000); Iwakiri Y, Satoh A, Chatterjee S, Toomre DK, Chalouni C M, Fulton D, Groszmann R J, Shah V H, Sessa W C. Proc. Natl. Acad. Sci. U S A 103:19777-19782 (2006)), and necessary for specific cell responses (see, e.g., Hess D T, Matsumoto A, Kim S O, Marshall H E, Stamler J S. Nat. Rev. Mol. Cell. Biol. 6:150-166 (2005)).

Aberrant or dysregulated denitrosylation or S-nitrosylation has been associated with stroke (cerebral ischemia) (see, e.g., Gu Z, Kaul M, Yan B, Kridel S J, Cui J, Strongin A, Smith J W, Liddington R C, Lipton S A. Science 297(5584):1186-90 (2002)) and a number of chronic degenerative diseases, including Parkinson's and Alzheimer's disease (see, e.g., Yao D, Gu Z, Nakamura T, Shi Z-Q, Ma Y, Gaston B, Palmer L A, Rockenstein E M, Zhang Z, Masliah E, Uehara T, Lipton S A. Proc. Natl. Acad. Sci. U S A 101(29):10810-4 (2004); Uehara T, Nakamura T, Yao D, Shi Z-Q, Gu Z, Masliah E, Nomura Y, Lipton S A. Nature 2441(7092):513-7 (2006); Benhar M, Forrester M T, Stamler J S. ACS Chem. Biol. 1(6):355-8 (2006); Cho D-H, Nakamura T, Fang J, Cieplak P, Godzik A, Gu Z, Lipton S A. Science 324(5923):102-5 (2009)) and Amyotrophic Lateral Sclerosis (ALS) (see, e.g., Schonhoff C M, et al. Proc. Natl. Acad. Sci. U S A 103(7):2404-9 (2006)). In addition, there is an emerging role of S-nitrosylation in cancer biology (see, e.g., Aranda E, López-Pedrera C, De La Haba-Rodriguez J R, Rodriguez-Ariza A. Curr. Mol. Med. 12(1):50-67 (2012); Aranda E, López-Pedrera C, De La Haba-Rodriguez J R, Rodriguez-Ariza A. Curr. Mol. Med. 12(1):50-67 (2012)).

S-nitrosylation has not yet been used within diagnostic and/or therapeutic methods. Yet, research publications on the topic increased from 69 in 2000 to 163 in 2012 according to Pubmed (CAGR 7%).

Current methods for detecting S-nitrosylation include several versions of the biotin switch technique, gold nanoparticle based enrichment, organomercury based methods, and phosphine-based probes. Limitation of biotin switch-based method is high false identification rate, due to mainly three reasons. First, the efficiency/sensitivity of this assay relies on complete blocking of reduced cysteine residues. Second, the efficiency of ascorbate reduction has been questioned. Third, there is the possibility of disulfide exchange after ascorbate reduction. Other methods for detecting S-nitrosylation include gold nanoparticle based enrichment, organomercury based methods, and phosphine-based probes. Limitations for gold nanoparticle based enrichment include, for example, that AuNPs react with both S-nitrosylated and S-glutathionylated cysteines, providing a challenge for absolute assignment of specific post-translational modifications to these residues. Phosphine based probes have yet to be evaluated in biological systems and organomercury approaches may be toxic.

The present invention overcomes such limitations, and provides new chemoselective ligation methods for the detection of protein S-nitrosylation. Indeed, experiments conducted during the course of developing embodiments for the present invention provide novel S-nitrosocysteine specific mild ligation approaches to directly label sites of proteins S-nitrosylation. In some embodiments, such approaches enable a one-step, direct covalent enrichment of SNO modified proteins with no cross-reactivity towards other cysteine post-translational modifications (PTMs).

In particular, such experiments determined that S-nitrosocysteine reacts with sulfinic acids in water at ambient temperature to form stable sulfonothioates and that this adduct is stable for flash chromatography, HPLC, and mass spectrometry analysis (see, FIG. 26). The cross-reactivity of the sulfinic acid with various thiol modifying reagents and other thiol modifications using an HPLC assay was next determined (Table 1). It was found that sulfinic acids react quickly and selectively with S-nitrosocysteines, but do not react with free thiols or disulfides. The sulfonothioate adduct was susceptible to thiol exchange with free cysteine, but this is prevented by pre-alkylation with iodoacetamide, but not maleimide.

Based on such findings, a strategy was devised to link the sulfinic acid group to a fluorescent dye or a biotin label. The fluorescent probe (e.g., sulfinic acid probe) was synthesized in good yield (˜73% isolated) from fluorescein NHS-ester and hypotaurine and purified on a preparative HPLC. Whether the probe could label S-nitrosylated proteins in cultured cancer cell lysates in the presence of NO donor (MAHMA-NONOate) was next tested (see, FIG. 27). After incubation, the protein was analyzed by SDS-PAGE and analyzed by in-gel fluorescence. Addition of the NO donor radically increased labeling, and pre-incubation with sodium ascorbate eliminated nearly all labeling. This indicated NO-dependent protein labeling.

Accordingly, in some embodiments, the present invention provides a selective, one-step SNO detection technique. The present invention is not limited to particular methods for SNO detection. In some embodiments, compositions comprising sulfinic acid are provided. In some embodiments, such compositions are exposed to cell samples having proteins with cysteine residues such that the sulfinic acid binds with such cysteine residues having undergone S-nitrosolyation. In some embodiments, the sulfinic acid binds with such S-nitrosothiol moieties resulting in a sulfonothioate adduct. In some embodiments, generation of a protein with a sulfonothioate adduct moiety following exposure to sulfinic acid indicates that such cysteine residue associated with a particular protein had undergone S-nitrosylation. In some embodiments, the protein can subsequently be separated from the sample and identified.

These probes efficiently labeled endogenous S-nitrosylated proteins, providing a direct method to covalently label, enrich, and annotate S-nitrosylated cysteines in cell and tissue lysates. Enrichment and mass spectrometry studies using biotin-hypotaurine (biotin-SO₂H) led to the identification of nearly 1000 endogenous S-nitrosylated proteins under basal conditions in cultured mammalian cells, providing the most sensitive and in-depth analysis of S-nitrosylation to date (see, FIG. 16). When compared with native protein abundance, a subset of S-nitrosylated proteins were found with enhanced stoichiometry, indicating functional enrichment. In addition, it was shown that this reaction is bi-directional, and S-nitrosothiol probes directly label S-sulfinylated proteins, such as the S-sulfinylated protein DJ-1.

As such, in certain embodiments, the present invention provides methods for detecting protein S-nitrosylation within thiol groups in proteins, metabolites, or materials. In particular, the present invention provides methods for detecting protein S-nitrosylation within cysteine residues of a protein, comprising providing a biological sample comprising proteins having cysteine residues and a composition comprising a labeled sulfinic acid moiety (e.g., a sulfinic acid probe), exposing the composition to the biological sample such that the labeled sulfinic acid moiety is able to interact with the nitrosothiols on cysteine side chains of the protein so as to generate labeled sulfonothioate moieties, and characterizing the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-nitrosylation. In some embodiments, the methods further comprise identifying the protein having been characterized as having undergone S-nitrosylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-nitrosylation.

In certain embodiments, the present invention further provides methods for detecting proteins having cysteine residues having undergone S-sulfinylation. Protein S-sulfinylation describes the oxidation of cysteine thiols to a SOOH motif via a peroxide-mediated pathway.

The present invention is not limited to particular methods for detecting proteins having cysteine residues having undergone S-sulfinylation. In some embodiments, such methods involve detecting proteins having cysteine residues having undergone S-sulfinylation through, for example, exposing such cysteine residues to a composition comprising maleimide in aqueous buffers at ambient temperature to provide a stable and detectable sulfone adduct (see, FIG. 28). In some embodiments, such methods involve detecting proteins having cysteine residues having undergone S-sulfinylation through, for example, exposing such cysteine residues to a composition comprising a labeled nitrosothiol moiety (e.g., biotin-GSNO). In some embodiments, generation of a protein with a sulfone adduct moiety following exposure to a composition comprising maleimide indicates that such cysteine residues associated with a particular protein had undergone S-sulfinylation. In some embodiments, the protein can subsequently be separated from the sample and identified. In some embodiments, the maleimide is labeled with an imaging agent (e.g., a fluorescent dye) to facilitate detection of the sulfone adduct. In some embodiments, such methods for S-sulfinylation detection may be used within in vivo samples, ex vivo samples, and/or in vitro samples.

The methods for S-sulfinylation detection are not limited to utilizing a particular type or kind of detection technique for detecting generation of a sulfone adduct moiety associated with a cysteine residue for a particular protein following exposure to a composition comprising maleimide in aqueous buffer (thereby indicating that such protein had undergone S-sulfinylation). In some embodiments wherein the maleimide is labeled with a fluorescent dye, in-gel fluorescence techniques are used. In some embodiments, flash chromatography is used to detect generation of a sulfone adduct moiety associated with a cysteine residue for a particular protein following exposure to a composition comprising maleimide in aqueous buffers. In some embodiments, high performance liquid chromatography (HPLC) is used to detect generation of a sulfone adduct moiety associated with a cysteine residue for a particular protein following exposure to a composition comprising maleimide in aqueous buffers. In some embodiments, mass spectrometry is used to detect generation of a sulfone adduct moiety associated with a cysteine residue for a particular protein following exposure to a composition comprising maleimide and aqueous buffer. In some embodiments, such methods are used to identify pharmaceutical agents (e.g., compounds, medicaments) capable of inhibiting or facilitating S-sulfinylation. In some embodiments, such identified pharmaceutical agents are used in the treatment of a disorder characterized by aberrant S-sulfinylation.

In certain embodiments, the present invention provides methods for detecting protein S-sulfinylation within cysteine residues of a protein, comprising providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled nitrosothiol moiety, exposing the composition to the biological sample such that the labeled nitrosothiol moiety is able to interact with sulfinic acid side chains of the cysteine residues so as to generate labeled sulfonothioate moieties, characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation. In some embodiments, the methods further comprise identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation.

In some embodiments, the labeled nitrosothiol moiety is a labeled nitrosoglutathione moiety. In some embodiments, the labeled nitrosothiol moiety is a labeled S-Nitroso-N-acetylpenicillamine moiety.

In some embodiments, the labeled sulfinic acid moiety comprises a labeling agent and/or an enrichment agent. In some embodiments, the labeled nitrosothiol moiety comprises a labeling agent and/or an enrichment agent. The methods for nitrosylation detection and sulfinylation detection are not limited to utilizing a particular type or kind of labeling agent. In some embodiments, the labeling agent is a fluorescent dye. Examples of such imaging agents include, but are not limited to, molecular dyes, fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and cis-parinaric acid. In some embodiments, the imaging agents are molecular dyes from the alexa fluor (Molecular Probes) family of molecular dyes. For example, examples of imaging agents include, but are not limited to, Alexa Fluor 350 (blue), Alexa Fluor 405 (violet), Alexa Fluor 430 (green), Alexa Fluor 488 (cyan-green), Alexa Fluor 500 (green), Alexa Fluor 514 (green), Alexa Fluor 532 (green), Alexa Fluor 546 (yellow), Alexa Fluor 555 (yellow-green), Alexa Fluor 568 (orange), Alexa Fluor 594 (orange-red), Alexa Fluor 610 (red), Alexa Fluor 633 (red), Alexa Fluor 647 (red), Alexa Fluor 660 (red), Alexa Fluor 680 (red), Alexa Fluor 700 (red), Alexa Fluor 750 (red), fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, cis-parinaric acid, Hoechst 33342, Brilliant Violet™ 421, BD Horizon™ V450, Pacific Blue™, AmCyan, phycoerythrin (PE), Brilliant Violet™ 605, BD Horizon™ PE-CF594, PI, 7-AAD, allophycocyanin (APC), PE-Cy™5, PerCP, PerCP-Cy™5.5, PE-Cy™7, APC-Cy7, BD APC-H7, Texas Red, Lissamine Rhodamine B, X-Rhodamine, TRITC, Cy2, Cy3, Cy3B, Cy3.5, Cy5.5, Cy7, BODIPY-FL, FluorX™, TruRed, Red 613, NMD, Lucifer yellow, Pacific Orange, Pacific Blue, Cascade Blue, Methoxycoumarin, coumarin, hydroxycoumarin, aminocoumarin, 3-azidocoumarin, DyLight 350, DyLight 405, DyLight 488, DyLight® 550, DyLight 594, DyLight 633, DyLight® 650, DyLight 680, DyLight 755, DyLight 800, Tracy 645, Tracy 652, Atto 488, Atto 520, Atto 532, Atto Rho6G, Atto 550, Atto 565, Atto 590, Atto 594, Atto 633, Atto Rho11, Atto Rho14, Atto 647, Atto 647N, Atto 655, Atto 680, Atto 700, CF™350, CF™405S, CF™405M, CF™488A, CF™543, CF™555, CF™568, CF™594, CF™620R, CF™633, CF™640R, CF™647, CF™660, CF™660R, CF™680, CF™680R, CF™750, CF™770, and CF™790. In some embodiments, the imaging agent is a mass-spec label selected from the group consisting of 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 156Gd, 158Gd, 159Tb, 160Gd, 162Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 174Yb, 175Lu, and 176Yb.

The methods for nitrosylation detection and sulfinylation detection are not limited to utilizing a particular type or kind of enrichment agent. In some embodiments, the enrichment agent is biotin and/or desthiobiotin for affinity enrichment.

The methods for nitrosylation detection and sulfinylation detection are not limited to utilizing a particular type or kind of detection technique for detecting generation of a labeled sulfonothioate moiety (thereby indicating that such protein had undergone S-nitrosylation or S-sulfinylation).

In some embodiments wherein the labeling agent is a fluorescent dye, in-gel fluorescence techniques are used to detect a labeled sulfonothioate moiety associated with a cysteine residue for a particular protein following exposure to either a labeled sulfinic acid moiety or a labeled nitrosothiol moiety.

In some embodiments, flash chromatography followed by nuclear magnetic resonance (NMR) analysis and/or mass spectrometry is used to detect generation of a labeled sulfonothioate moiety associated with a cysteine residue for a particular protein following exposure to either a labeled sulfinic acid moiety or a labeled nitrosothiol moiety.

In some embodiments, high performance liquid chromatography (HPLC) coupled with by mass spectrometry is used to detect generation of a labeled sulfonothioate moiety associated with a cysteine residue for a particular protein following exposure to either a labeled sulfinic acid moiety or a labeled nitrosothiol moiety.

In some embodiments, mass spectrometry used to detect a labeled sulfonothioate moiety associated with a cysteine residue for a particular protein following exposure to either a labeled sulfinic acid moiety or a labeled nitrosothiol moiety. In some embodiments, the sulfonothioate moiety associated with a cysteine residue for a particular protein following exposure to either a labeled sulfinic acid moiety or a labeled nitrosothiols moiety is cleaved by TCEP to generate either a free thiol or a sulfinic acid on the cysteine residue. Mass spectroscopy is used to identify the newly generated sulfinic acid and is also used to identify the newly generated free thiol, which is capped by N-ethyl maleimide. This additional manipulation permits annotation of the site of nitrosylation or sulfinylation in the protein.

As used herein, the term “flash chromatography” means the separation of mixtures by passing a fluid mixture dissolved in a “mobile phase” under pressure through a column comprising a stationary phase, which separates the analyte (i.e., the target substance) from other molecules in the mixture and allows it to be isolated (see, e.g., J. Org. Chem. 1978, 43,2923).

“Mass spectrometry,” as used herein, refers to a method comprising employing an ionization source to generate gas phase ions from a biological entity of a sample presented on a biologically active surface, and detecting the gas phase ions with an ion detector. Comparison of the time the gas phase ions take to reach the ion detector from the moment of ionization with a calibration equation derived from at least one molecule of known mass allows the calculation of the estimated mass to charge ratio of the ion being detected. The term “mass spectrometer” refers to a gas phase ion spectrometer that includes an inlet system, an ionization source, an ion optic assembly, a mass analyzer, and a detector.

As used herein, the term “nuclear magnetic resonance (NMR) signal” is intended to mean an output representing the frequency of energy absorbed by a population of magnetically equivalent atoms in a magnetic field, the magnitude of energy absorbed at the frequency by the population and distribution of frequencies around a central frequency. The frequency of energy absorbed by with an atom in a magnetic field can be determined from the location of a peak in an NMR spectrum. The magnitude of energy absorbed at a frequency by a population of atoms can be determined from relative peak intensity. The distribution of frequencies around a central frequency can be determined from the shape of a peak in an NMR spectrum. Accordingly, a collection of nuclear magnetic resonance signals for a molecule or sample containing multiple atoms can be represented in an NMR spectrum, as an atom having a signal of characteristic frequency, intensity and line-shape.

As used herein, the term “high performance liquid chromatography” or “HPLC” (sometimes known as “high pressure liquid chromatography”) refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

In certain embodiments, the present invention provides methods for identifying proteins having undergone S-nitrosylation and/or to annotate/profile particular locations of such S-nitrosylation. In certain embodiments, the present invention provides methods for identifying proteins having undergone S-sulfinylation and/or to annotate/profile particular locations of such S-sulfinylation. For example, in some embodiments, complex cell lysates or tissue samples are treated with a labeled sulfinic acid moiety or a labeled nitrosothiol moiety, such treated samples treated are prepared for mass spectrometric analysis, and peptidic fragments are separated by liquid chromatography (LC) followed by mass spectrometry. In some embodiments, the methods further involve proteomic analysis using bioinformatics and the raw data is searched against, for example, a defined database to identify particular proteins and to detect peptides bearing the sulfonothioate adduct (or the corresponding mass reporter ion) to annotate and profile sites of labeling. Indeed, experiments conducted during the course of developing embodiments for the present invention, through enrichment and mass spectrometry studies using biotin-hypotaurine (biotin-SO₂H) led to the identification of nearly 1000 endogenous S-nitrosylated proteins under basal conditions in cultured mammalian cells, providing the most sensitive and in-depth analysis of S-nitrosylation to date (see, FIG. 16). In addition, when compared with native protein abundance, a subset of S-nitrosylated proteins were found with enhanced stoichiometry, indicating functional enrichment.

The methods for nitrosylation detection and sulfinylation detection are not limited to particular uses. In some embodiments, such methods for nitrosylation detection and sulfinylation detection are used within biological settings. For example, in some embodiments, the methods for nitrosylation detection are used to screen biological samples for the presence of nitrosothiols. In some embodiments, the methods for nitrosylation detection are used to detect the presence of nitrosothiols within a particular biological sample (e.g., a biological sample from a patient (e.g., a clinical biopsy)).

In some embodiments, such methods for nitrosylation detection and sulfinylation detection may be used within in vivo samples, ex vivo samples, and/or in vitro samples.

In some embodiments, such methods for nitrosylation detection and sulfinylation detection are used to assist in characterizing disorders (e.g., strokes, chronic degenerative diseases, including Parkinson's and Alzheimer's disease; Amyotrophic Lateral Sclerosis (ALS); cancer) involving the occurrence of protein S-nitrosylation (e.g., through SNO detection) (e.g., through quantification of SNO presence).

In some embodiments, such methods for nitrosylation detection are used to identify pharmaceutical agents (e.g., compounds, medicaments) capable of preventing S-nitrosylation. The present invention is not limited to particular methods for identifying pharmaceutical agents capable of preventing S-nitrosylation. In some embodiments, identification of pharmaceutical agents capable of preventing S-nitrosylation involves, for example, exposing a sample having proteins having cysteine residues known to undergo S-nitrosylation to a pharmaceutical agent, detecting the presence or absence of S-nitrosylation with the methods of the present invention, and identifying such pharmaceutical agent as being capable of preventing S-nitrosylation if such methods are unable to detect S-nitrosylation. In some embodiments, pharmaceutical agents identified as S-nitrosylation inhibitors are further characterized with regard to its inhibitory effect on specific proteins. In some embodiments, such pharmaceutical agents identified as S-nitrosylation inhibitors are used in methods for treating subjects (e.g., human patients) suffering from disorders involving the aberrant occurrence of protein S-nitrosylation (e.g., chronic degenerative diseases, including Parkinson's and Alzheimer's disease; Amyotrophic Lateral Sclerosis (ALS); cancer).

In some embodiments, such methods for nitrosylation detection are used to identify pharmaceutical agents (e.g., compounds, medicaments) capable of facilitating S-nitrosylation. The present invention is not limited to particular methods for identifying pharmaceutical agents capable of facilitating S-nitrosylation. In some embodiments, identification of pharmaceutical agents capable of facilitating S-nitrosylation involves, for example, exposing a sample having proteins having cysteine residues known to not undergo S-nitrosylation to a pharmaceutical agent, detecting the presence or absence of S-nitrosylation with the methods of the present invention, and identifying such pharmaceutical agent as being capable of facilitating S-nitrosylation if such methods are able to detect S-nitrosylation. In some embodiments, pharmaceutical agents identified as S-nitrosylation facilitators are further characterized with regard to its facilitating effect on specific proteins. In some embodiments, such pharmaceutical agents identified as S-nitrosylation facilitators are used in methods for treating subjects (e.g., human patients) suffering from disorders involving the aberrant occurrence of protein S-nitrosylation.

In some embodiments, such methods for sulfinylation detection are used to identify pharmaceutical agents (e.g., compounds, medicaments) capable of preventing S-sulfinylation. The present invention is not limited to particular methods for identifying pharmaceutical agents capable of preventing S-sulfinylation. In some embodiments, identification of pharmaceutical agents capable of preventing S-sulfinylation involves, for example, exposing a sample having proteins having cysteine residues known to undergo S-sulfinylation to a pharmaceutical agent, detecting the presence or absence of S-sulfinylation with the methods of the present invention, and identifying such pharmaceutical agent as being capable of preventing S-sulfinylation if such methods are unable to detect S-sulfinylation. In some embodiments, pharmaceutical agents identified as S-sulfinylation inhibitors are further characterized with regard to its inhibitory effect on specific proteins. In some embodiments, such pharmaceutical agents identified as S-sulfinylation inhibitors are used in methods for treating subjects (e.g., human patients) suffering from disorders involving the aberrant occurrence of protein S-sulfinylation.

In some embodiments, such methods for sulfinylation detection are used to identify pharmaceutical agents (e.g., compounds, medicaments) capable of facilitating S-sulfinylation. The present invention is not limited to particular methods for identifying pharmaceutical agents capable of facilitating S-sulfinylation. In some embodiments, identification of pharmaceutical agents capable of facilitating S-sulfinylation involves, for example, exposing a sample having proteins having cysteine residues known to not undergo S-sulfinylation to a pharmaceutical agent, detecting the presence or absence of S-sulfinylation with the methods of the present invention, and identifying such pharmaceutical agent as being capable of facilitating S-sulfinylation if such methods are able to detect S-sulfinylation. In some embodiments, pharmaceutical agents identified as S-sulfinylation facilitators are further characterized with regard to its facilitating effect on specific proteins. In some embodiments, such pharmaceutical agents identified as S-sulfinylation facilitators are used in methods for treating subjects (e.g., human patients) suffering from disorders involving the aberrant occurrence of protein S-sulfinylation.

EXPERIMENTAL Example I

While exploring the interplay of cysteine post-translational modifications, a reported reaction between phenylsulfinic acid and S-nitrosocysteine was identified, leading to thiosulfonate formation in aqueous buffers at room temperature (see, e.g., Hart, T. W. Tetrahedron Letters 26, 2013-2016 (1985)) (FIG. 1 a, FIG. 2). Thiosulfonates are readily exchangeable with thiols, serving as the basis for the cysteine capping agent methyl methanethiosulfonate (MMTS). To prevent such exchange, it was found that sulfinic acids do not react with iodoacetamide, enabling orthogonal alkylation of thiols without perturbing nitrosothiols or sulfinic acids (FIG. 3). Furthermore, it was found that sulfinic acids do not react with thiols (cysteine), disulfides (cystine or 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)), or aldehydes (benzaldehyde) (FIG. 4). Additionally, the thiosulfonate product is unaffected by ascorbate, but reduced by tris(2-carboxyethyl)phosphine (TCEP) (FIG. 5). Most S-sulfinylation studies alkylate thiols with MMTS (see, e.g., Doulias, P. T. et al. Sci Signal 6, rs1 (2013); Forrester, M. T., Foster, M. W. & Stamler, J. S. of Biol Chem 282, 13977-83 (2007); Jaffrey, S. R. et al. Nat Cell Biol 3, 193-7 (2001)), which reacts with cysteine to release methylsulfinic acid. Based on our findings, methylsulfinic acid will react with S-nitrosothiols to form a thiosulfonate, which can then catalyze disulfide formation with free thiols and hamper S-sulfinylation detection.

Thiosulfonate formation was further characterized by measuring the loss of S-nitroso-glutathione (GSNO) absorbance after phenylsulfinic acid addition (FIG. 6). At pH 1 and 4, the reaction rate is first order and proceeds at 3×10⁻⁵ M.sec⁻¹. At neutral pH, a mild sigmoidal concentration dependence is observed, and no reaction occurs under basic conditions. Further LC-MS analysis revealed fractional formation of piloty's acid (N-hydroxybenzenesulfonamide) (FIG. 7). This is consistent with protonation of the nitrosothiol species, followed by addition by the phenylsulfinate anion to form a transient intermediate susceptible to attack by a second phenylsufinate anion to form thiosulfonate and piloty's acid (see, e.g,., Reeves, B. D. et al. Tetrahedron Lett 54 (2013)).

Next, the reactivity of sulfinic acids with native S-nitrosylated proteins was examined. Biotin and fluorescein N-hydroxysuccinimide (NHS) esters were directly coupled to the biological sulfinic acid metabolite hypotaurine (biotin-SO₂H) or the sulfonic acid metabolite taurine (biotin-SO₃H). Each probe was incubated with mammalian cell lysates pre-alkylated with excess iodoacetamide (FIG. 1b ) in 6 M urea/phosphate buffered saline (PBS), and separated by non-reducing SDS-PAGE for transfer to nitrocellulose and streptavidin detection. In contrast to biotin-SO₃H, biotin-SO₂H labeled a rich profile of proteins (FIG. 1c and FIG. 8) competed by excess hypotaurine (FIG. 9). Lysates treated with the NO donor methylamine hexamethylene methylamine NONOate (MAHMA-NONOate) showed increased labeling (FIG. 1d and FIG. 10), which was blocked by pre-treatment with ascorbate, and eliminated by post-incubation with TCEP (FIG. 11). Addition of the sulfenic acid blocking agent dimedone (see, e.g., Paulsen, C. E. & Carroll, K. S. Chem Rev 113, 4633-79 (2013); Benitez, L. V. & Allison, W. S. J Biol Chem 249, 6234-43 (1974)) had no effect on biotin-SO₂H conjugation (FIG. 1e ), suggesting little or no cross-reactivity. Importantly, dimedone labeling is largely eliminated in such denaturing conditions, as well as by ascorbate treatment (see, e.g., Reisz, J. A. et al. FEBS J 280, 6150-61 (2013)) (common to biotin-switch methods) (FIG. 12). Such reactivity is not unprecedented, and is used by the mitochondrial enzyme sulfide quinone oxidoreductase (SQR) in the conversion of hydrogen sulfide to a persulfide intermediate (SQR-SSH), which is then attacked by sulfinate to release thiosulfate (see, e.g., Jackson, M. R., Melideo, S. L. & Jorns, M. S. Biochemistry 51, 6804-6815 (2012)). Since all thiols are completely alkylated by continuous incubation with iodoacetamide (FIG. 13), persulfidation (R-SSH) is constitutively blocked (see, e.g., Pan, J. & Carroll, K. S. ACS Chem Biol 8, 1110-6 (2013)), providing an orthogonal workflow for selective conjugation and detection of endogenous S-nitrosothiols.

Given the efficient and selective conjugation of sulfinic acids and nitrosothiols, recombinant human GAPDH were next purified for targeted analysis of S-sulfinylation with biotin-SO₂H. Detection of GAPDH S-sulfinylation was enhanced by nitric oxide donors, unaffected by dimedone, and slightly diminished by addition of hydrogen peroxide (FIG. 14), likely caused by further oxidation and inactivation of the probe. Biotin-SO₂H-labeled GAPDH protein was digested with trypsin and analyzed by high-resolution mass spectrometry, identifying a biotin-thiosulfonate modified peptide encompassing the GAPDH catalytic cysteine (FIG. 15). Interestingly, MS/MS analysis identified S-sulfinylation of Cys156, and not the catalytic nucleophile Cys152 that is reported to mediate trans-sulfinylation to other targets (see, e.g., Kornberg, M. D. et al. Nat Cell Biol 12, 1094-100 (2010)). These studies confirm direct detection of native of S-sulfinylation by sulfinic acid probes for mass spectrometry analysis.

Next, biotin-SO₂H labeling was combined with stable-isotope labeling with amino acids in cell culture (SILAC) for quantitative mass spectrometry annotation of endogenous S-sulfinylation in mammalian cells. Heavy or light cell 293T cell lysates were separately alkylated with excess iodoacetamide in denaturing buffers, followed by incubation with biotin-SO₂H or biotin-SO₃H, respectively. After chloroform/methanol precipitation, the two lysates were combined for streptavidin enrichment, trypsin digestion, and mass spectrometry analysis using an in-line fractioning column for multidimensional analytical separation, electrospray nanoLC, and high-resolution analysis by a quadrupole ion mobility time-of-flight mass spectrometer. Peptides were analyzed using data-independent acquisition methods in combination with ion mobility separation (HDMS^(E)) and mobility-dependent collision energy assignment for enhanced fragmentation (see, e.g., Distler, U. et al. Nat Methods (2013)). Through a combination of 4 biological replicates, each with 2 technical replicates, a total of 992 proteins were identified with SILAC ratios >5 (biotin-SO₂H/biotin-SO₃H), quantified in ≧3 replicates, and represented by ≧3 quantified peptides (FIG. 16). This list includes nearly all previously annotated S-nitrosylated proteins, including ion channels, chaperones, peroxiredoxins, p53, HDACs, hundreds of metabolic enzymes, as well as a rich set of novel proteins. Site-specific profiling of endogenous S-sulfinylation will require additional adaptations, since dithionite used to reduce azobenzene cleavable linkers (see, e.g., Yang, Y. Y. et al. Chem Biol 17, 1212-22 (2010)) also reduces thiosulfonates, and release by thiosulfonate reduction will also reduce latent disulfides.

Matched unenriched cell lysates were digested with trypsin for mass spectrometry analysis, and the relative abundance of ˜3000 proteins were determined by label-free quantitative methods (FIG. 17). Approximately 500 S-nitrosylated proteins were quantified in the unenriched analysis. Comparison of gene ontology identifiers in both datasets showed little functional enrichment across protein classes (see, e.g., Huang da, W., Sherman, B. T. & Lempicki, R. A. Nat Protoc 4, 44-57 (2009)), except an increased representation of S-nitrosylated chaperone proteins (FIG. 18).

Next, S-sulfinylation enrichment was qualified in comparison with native abundance to identify S-nitrosylated proteins with particularly high stoichiometry. Label-free absolute quantification was performed for each dataset using the top three ionized tryptic peptides, which is directly proportional to protein abundance over a wide dynamic range (see, e.g., Silva, J. C. et al., Molecular & Cellular Proteomics 5, 144-156 (2006); Ahrne, E., Molzahn, L., Glatter, T. & Schmidt, A. Proteomics 13, 2567-78 (2013)). Individual protein values from the biotin-SO₂H enrichment were divided by their corresponding abundance observed in the unenriched analysis, providing a distribution of ratios reflecting proportionally higher S-sulfinylation occupancy (FIG. 18 and FIG. 19). The majority of proteins were observed with low ratios, signifying poor relative enrichment and low S-sulfinylation stoichiometry, including several heat shock proteins, tubulin, and ribosomal proteins. In contrast, S-nitrosylated proteins with large ratios signify high stoichiometry, including several metabolic enzymes and proteins with metal coordination sites, such as HDAC1 and carbonic anhydrase. While only half of the putative S-nitrosylated proteins assigned in our enrichment were assessed in this comparison, we can infer that basal nitrosative damage fractionally modifies many abundant proteins, while a subset of proteins demonstrate higher native S-sulfinylation occupancy. Understandably, S-sulfinylation is presumably amplified in cells cultured in atmospheric oxygen, warranting further analysis under physiological oxygen conditions.

It was next asked what would happen if the detection scheme was reversed, using S-nitrosothiol-linked probes to detect endogenous S-sulfinylation. Similar reactivity was recently reported using aryl-nitroso ligation to several sulfinic acid standards, including glutathione, to form a stable N-sulfonylbenzisoxazolone (see, e.g., Lo Conte, M. & Carroll, K. S. Angew Chem Int Ed Engl 51, 6502-5 (2012)). Despite such progress, there are no reported methods to selectively profile endogenous S-sulfinylation in complex proteomes. In order to test this approach, recombinant human DJ-1 was purified, a redox chaperone that spontaneously forms a stable sulfinic acid at Cys106 (see, e.g., Canet-Aviles, R. M. et al. Proc Natl Acad Sci U S A 101, 9103-8 (2004)). After iodoacetamide alkylation of free thiols, N-acetyl-S-nitrosocysteine methyl ester was added to DJ-1, and processed for high-resolution LC-MS analysis (FIG. 20A and FIG. 21)). MS/MS analysis unambiguously confirmed thiosulfonate formation at Cys106-SO₂H (FIG. 20B). Furthermore, there were no detectable N-hydroxysulfonamide cysteine modifications, implying one sulfinic can be sufficient for thiosulfonate formation. Next, biotin-GSNO was synthesized in one step from biotin-NHS and GSNO, and added to mammalian lysates denatured in 6 M urea/PBS and pre-alkylated with iodoacetamide. Gel-based analysis revealed a distinct profile of putative S-sulfinylated proteins (FIG. 3C), validating the intrinsic cross-reactivity nitrosothiols and sulfinic acids. Because these probes are particularly light sensitive, probe synthesis and purification immediately preceded proteome labeling. Future efforts will apply these methods for selective profiling of proteome-wide dynamics of S-sulfinylation, and develop new orthogonal strategies based on the sulfinic acid nucleophilicity.

In summary, by harnessing the inherent cross-reactivity between sulfinic acids and nitrosothiols we demonstrate bi-directional profiling of native cysteine modifications. While the rate of this reaction is relatively slow in solution, certain S-sulfinylated enzymes may accelerate thiosulfonate formation, and could promote exchange with cellular thiols to regenerate the sulfinic acid. Overall, these findings establish that sulfinic acids are nucleophilic, and possess intrinsic reactivity that may contribute to cellular redox regulation.

Example II

This example describes synthetic methods pertaining to Example I.

All compounds were purchased from Sigma-Aldrich, unless otherwise noted. NMR analysis was performed using a Varian 400 MHz NMR instrument. Small molecule high-resolution mass spectrometry was performed using an electrospray Agilent Q-TOF mass spectrometer (accuracy 1-5 ppm). Low-resolution mass spectrometry was performed using an electrospray Micromass LCT time-of-flight mass coupled to a HPLC pump with a rheodyne loop injector. Compounds were purified by normal phase silica column chromatography or by semi-prep High-Performance Column Chromatography (HPLC). HPLC purifications were performed using a Waters semi-preparative 1525 binary pump system coupled to a photodiode array detector, an autosampler, and an automatic fraction collector. Separations were carried out on an Atlantis prep T3 C₁₈ column (10×250 mm), in 95/5 water/acetonitrile 0.1% formic acid for 2 minutes, followed by a 40 minute gradient increasing the mobile phase to 5/95 water/acetonitrile with 0.1% formic acid. Data were analyzed using Waters Empower software. Resulting HPLC fractions were lyophilized using a Labconco FreeZone2.5-Plus freeze-drying system.

(R)-methyl 2-acetamido-3-(nitrosothio)propanoate (2). The nitrosothiol was synthesized based on the method reported earlier¹. Briefly, an amber round bottom flask was charged with N-acetyl-L-cysteine methyl ester (1, 1 eq., 100 mg, 0.56 mmol). The contents were dissolved in methanol (3 mL) and 1 N hydrochloric acid (2 mL), and cooled to 0° C. for 15 minutes. An ice-cold solution of sodium nitrite (1.1 eq., 42.8 mg, 0.62 mmol) in water (1 mL) was then slowly added to the solution of N-acetyl-L-cysteine methyl ester in hydrochloric acid and methanol. The reaction mixture was constantly kept in the dark at 0° C., and allowed to proceed for 30 minutes. HPLC analysis of the crude reaction mixture was performed to monitor completion of the reaction. The crude product was then transferred to an amber reparatory funnel and extracted with ethyl acetate (3×2 mL). The combined organic extracts were pooled, dried with sodium sulfate (200 mg), filtered and evaporated under vacuum (in the absence of light). This afforded a reddish-pink residue, which was used directly for subsequent reactions within 30 minutes. An HPLC trace of both the starting materials and the S-nitroso product is shown in FIG. 22 (HPLC trace of N-acetyl-cysteine-OMe starting material and the S-nitroso product formed upon reaction with sodium nitrite in 1 N HCl and methanol). (R)-methyl 2-acetamido-3-((phenylsulfonyl)thio)propanoate (3). Thiosulfonate compound 3 was synthesized based on a method reported earlier^(1,2). Briefly, the N-acetyl-S-nitrosocysteine methyl ester, 2, obtained in reaction above was dissolved in water followed by addition of sodium benzenesulfinate (3 eq., 1.68 mmol, 275.8 mg). The contents were allowed to react for 3 hours at ambient temperature in darkness. The contents were then transferred to a separatory funnel and extracted with ethyl acetate (3×10 mL). The organic fractions were pooled, washed with brine (10 mL), dried over sodium sulfate (500 mg), and concentrated under vacuum to afford an oily residue, which was further purified by flash column chromatography (isocratic elution 5% methanol in methylene chloride). This yielded 178 mg of thiosulfonate product 3. ¹H NMR (400 MHz, CDCl₃) δ 7.91-7.85 (m, 2H), 7.66-7.59 (m, 1H), 7.57-7.51 (m, 2H), 6.60 (d, J=7.5 Hz, 1H), 4.81 (dt, J=7.5, 5.1 Hz, 1H), 3.71 (s, 3H), 3.54-3.36 (m, 2H), 1.99 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.32, 169.94, 144.02, 134.11, 129.49, 127.06, 53.05, 51.59, 37.08, 22.96. LR-ESI (Pos): m/z=318.04 [M+H]⁺. FIG. 23 shows spectra of (a) ¹H NMR of compound 3. (b) ¹³C NMR of compound 3.

2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((2-sulfinoethyl)carbamoyl)benzoic acid (5). A scintillation vial was charged with a solution of NHS-fluorescein, 4 (Pierce, 1 eq., 7 mg, 0.015 mmol) in degassed THF (1 mL), and the contents were allowed to cool to 0° C. The atmosphere in the flask was replaced with nitrogen. A solution of hypotaurine (3.0 eq., 4.8 mg, 0.045 mmol) in degassed (sonication, 3 freeze-thaw cycles) 0.01N sodium hydroxide (100 μL) was then added to the NHS-flourescein. The contents were allowed to react for 4 hours under an atmosphere of nitrogen and progress was monitored by HPLC and mass spectrometry (LR-ESI-Pos). The product, 5, was purified to homogeneity by semi-preparative HPLC and lyophilized to yield compound 5 as a bright yellow solid in 29% yield. HRMS (ESI Negative), [M-H]⁻ Calculated: m/z=466.0602, Found: m/z=466.0570. 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((2-sulfoethyl)carbamoyl)benzoic acid (6): A scintillation vial was charged with a solution of NHS-fluorescein, 4, (Pierce, 1 eq., 15 mg, 0.032 mmol) in THF and the contents were allowed to cool to 0° C. A solution of taurine (3 eq., 11.9 mg, 0.095 mmol) in 0.01N sodium hydroxide (100 μL) was then added to the NHS-flourescein. The contents were allowed to react for 10 hours and progress was monitored by HPLC and mass spectrometry (LR-ESI-Pos). The product was purified to homogeneity by semi-preparative HPLC and then lyophilized to afford compound 6 as a bright yellow solid in 55% yield. HRMS (ESI Positive), [M+H]⁺ Calculated: m/z=484.0697, Found: m/z=484.0693.

2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl)pentanamido) ethanesulfinic acid (8). A scintillation vial was charged with a solution of NHS-biotin, 7, (Pierce, 1.0 eq., 35 mg, 0.1 mmol) in a 1:1 mixture of degassed THF and water and the contents were allowed to cool to 0° C. A solution of hypotaurine (3 eq., 34 mg, 0.3 mmol) in degassed (sonication under vacuum followed by three freeze-thaw cycles under vacuum) 0.01 N sodium hydroxide (100 μL) was then added to the NHS-biotin. The contents were allowed to react for 2 hours and the reaction progress was monitored by HPLC and mass spectrometry (LR-ESI-Neg). The product was purified to homogeneity by semi-preparative HPLC and then lyophilized to afford compound 8 as a white solid in 34% yield. ¹H NMR (400 MHz, Deuterium Oxide) δ 4.44 (dd, J=7.9, 4.8 Hz, 1H), 4.26 (dd, J=7.9, 4.4 Hz, 1H), 3.35 (t, J=6.5 Hz, 2H), 3.17 (ddd, J=8.8, 5.8, 4.4 Hz, 1H), 2.83 (dd, J=13.0, 4.9 Hz, 1H), 2.68-2.45 (m, 2H), 2.09 (t, J=7.2 Hz, 2H), 1.63-1.31 (m, 4H), 1.32-1.11 (m, 2H). ¹³C NMR (100 MHz, D₂O) δ 176.71, 165.63, 61.94, 60.15, 58.43, 55.19, 39.57, 35.28, 33.12, 27.66, 25.07, 24.91. HRMS (ESI Negative), [M-H]⁻ Calculated: m/z=334.0901, Found: m/z=334.0896. FIG. 24 shows spectra of (a) Proton NMR of compound 8 (b) Carbon NMR of compound 8. 2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl)pentanamido) ethanesulfonic acid (9). A scintillation vial was charged with a solution of NHS-biotin, 7, (Pierce, 10 mg, 0.03 mmol) in a 1:1 mixture of THF and water and the contents were allowed to cool to 0° C. A solution of taurine (3 eq., 11 mg, 0.09 mmol) in 0.01 N sodium hydroxide (100 μL) was then added to the NHS-biotin. The contents were allowed to react for 2 hours and progress was monitored by HPLC and mass spectrometry (LR-ESI-Pos). The product was purified to homogeneity by semi-preparative HPLC and lyophilized to afford compound 9 as white solid in 43% yield. ¹H NMR (400 MHz, Deuterium Oxide) δ 4.44 (dd, J=7.9, 4.8 Hz, 1H), 4.26 (dd, J=8.0, 4.5 Hz, 1H), 3.40 (t, J=6.8 Hz, 2H), 3.24-3.07 (m, 1H), 2.91 (t, J=6.7 Hz, 2H), 2.83 (dd, J=13.0, 4.9 Hz, 1H), 2.61 (d, J=13.2 Hz, 1H), 2.10 (t, J=7.4 Hz, 2H), 1.48 (dtt, J=26.3, 14.6, 7.1 Hz, 4H), 1.25 (q, J=7.6 Hz, 2H). HRMS (ESI Positive) [M+H]⁺ Calculated: m/z=352.0995, Found: m/z=352.0995.

5-hexyn-1-al (11): Compound 11 was synthesized as described previously³. Briefly, to a stirring solution of (2,2,6,6-tetramethylpiperidin-1-yl)oxy1 (TEMPO, 0.1 eq., 0.48 g, 3 mmol,) and (diacetoxyiodo)benzene (1.1 eq., 10.83 g, 33 mmol,) in anhydrous methylene chloride, 5-hexyn-1-ol (1 eq.,3.37 mL, 30 mmol,) was added drop-wise over the course of 30 minutes at ambient temperature and allowed to react for 3 hours. The reaction mixture was then transferred to a reparatory funnel and extracted with saturated sodium bicarbonate (2×20 mL) and brine (2×10 mL). The organic layer was then dried over anhydrous magnesium sulfate (1.5 g) and filtered. The solvent was removed under vacuum using a rotary evaporator the residue was impregnated onto a silica gel column and purified using flash column chromatography (gradient elution using 100% hexanes, 50% hexanes: 50% DCM and 100% DCM) to afford 1.907 g (64%) of compound 11 as a yellowish oil. ¹H NMR (400 MHz, Chloroform-d) δ 9.60 (s, 1H), 2.41 (td, J=7.2, 1.3 Hz, 2H), 2.07 (td, J=6.9, 2.6 Hz, 2H), 1.85 (t, J=2.7 Hz, 1H), 1.65 (p, J=7.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 201.46, 83.08, 69.29, 42.30, 20.66, 17.53. ethyl (E)-oct-2-en-7-ynoate (12): Compound 12 was synthesized by adapting a protocol described earlier⁴. Briefly, to stirring neat 5-hexyn-al (11, 1 eq., 0.50 g, 5 mmol,) triethyl phosphonoacetate (1.1 eq., 1.13 mL, 6 mmol,) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.5 eq., 1.17 mL, 8 mmol,) were added left for 12 hours at ambient temperature. The resulting reaction mixture was diluted with ethyl acetate, transferred to a reparatory funnel, and extracted with 1 M hydrochloric acid (2×20 mL) and brine (2×20 mL). The organic layer was collected and dried over anhydrous magnesium sulfate (500 mg), filtered, and dried under vacuum. The residue was impregnated onto a silica gel column and purified using flash column chromatography (isocratic elution using 1:1 hexanes : methylene chloride) to afford 806 mg (93%) of compound 12 as a yellowish oil. ¹H NMR (400 MHz, Chloroform-d) δ 6.86 (dtd, J=15.4, 7.0, 1.0 Hz, 1H), 5.78 (dt, J=15.6, 1.4 Hz, 1H), 4.11 (q, J=7.2, 1.0 Hz, 2H), 2.29-2.24 (m, 2H), 2.21 (dd, J=17.9, 1.2 Hz, 2H), 1.91 (td, J=2.7, 1.0 Hz, 1H), 1.68-1.54 (m, 2H), 1.24-1.18 (m, 3H). Synthesis of ethyl 2,4-dioxo-6-(pent-4-yn-1-yl)cyclohexane-1-carboxylate (13): Compound 13 was synthesized through minor modifications of a protocol described earlier (see, e.g., Focella, A., et al., J Organic Chemistry 42, 3456-3457 (1977)). Briefly, the atmosphere in a flame-dried round-bottom flask was replaced with dry nitrogen and the flask was allowed to attain ambient temperature. The cooled flask was charged with anhydrous methanol, followed by addition of ethyl (E)-oct-2-en-7-ynoate (1 eq., 5 mmol, 880 mg), and ethyl acetoacetate (3 eq., 16 mmol, 2.03 mL) and sodium methoxide (5 eq., 26 mmol, 1.43 g) were added. The mixture was refluxed under nitrogen for 6 hours, dried under vacuum, and extracted with methylene chloride (2×20 mL). The aqueous layer was acidified to pH 4 and extracted again with methylene chloride (2×20 mL). The organic layer was dried over anhydrous magnesium sulfate (500 mg), filtered, and the solvent removed under reduced pressure. The mixture was then purified by HPLC and lyophilized to yield 55 mg (32%) of compound 13 as a while solid (mixture of diastereomers). ¹H NMR (400 MHz, Chloroform-d) δ 3.79 (s, 3H), 3.78-3.66 (m, 2H), 3.12 (d, J=9.8 Hz, 1H), 3.08-2.98 (m, 1H), 2.53 (d, J=4.2 Hz, 1H), 2.14 (dtd, J=13.5, 6.3, 3.5 Hz, 2H), 1.91 (dt, J=10.4, 2.7 Hz, 2H), 1.46 (d, J=7.3 Hz, 2H), 1.31-1.14 (m, 2H). HRMS (ESI positive) [M+H]⁺: Calculated m/z: 237.1126, Found m/z=237.1118 5-(pent-4-yn-1-yl)cyclohexane-1,3-dione (14): A round bottom flask was charged with ethyl 2,4-dioxo-6-(pent-4-yn-1-yl)cyclohexane-1-carboxylate, 13, and 20 mL of 20% aqueous sodium hydroxide. The contents were allowed to react under reflux for 2 hours, and then cooled to ambient temperature, acidified with concentrated sulfuric acid, and refluxed for two additional hours. Next, the pH was adjusted to pH 4.0 and the reaction extracted with ethyl acetate. The organic layer was dried with magnesium sulfate (500 mg), filtered and the solvent removed under reduced pressure. The reaction mixture was purified by HPLC and lyophilized to yield 99% of compound 14 as a white solid. ¹H NMR (400 MHz, Chloroform-d) δ0 2.59-2.27 (m, 4H), 2.24-2.05 (m, 4H), 1.98-1.88 (m, 1H), 1.50 (dtt, J=21.4, 12.7, 5.0 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 178.03, 83.94, 68.60, 57.89, 47.49, 46.21, 37.98, 33.17, 30.27, 25.68, 18.40. HRMS (ESI positive) [M+H]⁺: Calculated m/z: 179.1072, Found m/z=179.1059.

Biotin-GSNO. 5-(((R)-1-((carboxymethyl)amino)-3-(nitrosothio)-1-oxopropan-2-yl) amino)-5-oxo-2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)pentanoic acid (16). A 25 mL scintillation vial was charged with 11 mg of Biotin-NHS ester (30 μmol, 1.1 eq), 10 mg S-nitroso-glutathione (Cayman, 30 μmol, 1 eq.), and 17 μL, triethylamine (119 μmol, 4 eq) in water/THF (2 mL) and stirred in the dark for 2 hours. The mixture was then purified by HPLC in the dark. The purified product (retention time 13 minutes) was lyophilized in the dark to obtain compound 16 in 43% yield. HRMS (ESI positive): Calculated m/z: [M+Na]⁺=585.1408, Found m/z [M+Na]⁺=585.1407. FIG. 25 shows HPLC trace of reaction mixture of biotin-GSNO, compound 16; Peak at minute 13.4 represents product as confirmed by MS analysis.

(S)-2-amino-5-(((R)-1-((carboxymethyl)amino)-1-oxo-3-(tosylthio)propan-2-yl) amino)-5-oxopentanoic acid (19): Compound 19 was synthesized based on a protocol described above². Briefly, to a cooled solution of S-nitroso-glutathione, 17, (GSNO, Cayman, 1.0 eq., 20 mg, 0.06 mmol) in degassed water, sodium 4-methyl-phenylsulfinate, 18 (3.0 eq., 32 mg, 0.18 mmol) was added allowed to react for 2 hours at ambient temperature in the dark. Compound 19 was purified from the crude reaction by semi-preparative HPLC. ¹H NMR (400 MHz, Methanol-d4) δ 7.87-7.76 (m, 1H), 7.69 (d, J=8.2 Hz, 1H), 7.43 (d, J=8.2 Hz, 1H), 7.21 (d, J=8.1 Hz, 1H), 4.68 (dd, J=8.1, 5.0 Hz, 1H), 3.90-3.84 (m, 2H), 3.79 (t, J=6.3 Hz, 1H), 3.41 (dd, J=14.1, 5.0 Hz, 1H), 3.26-3.21 (m, 1H), 2.50 (t, J=7.0 Hz, 2H), 2.44 (s, 3H), 2.35 (s, 2H), 2.17-2.07 (m, 2H). HRMS (ESI positive), [M+H]⁺ Calculated m/z: 461.0927, Found m/z=461.0924.

Example III.

This example describes materials and methods pertaining to Examples I and II.

Rate Constant Determination.

S-nitrosoglutathione (GSNO, Cayman) and sodium phenylsulfinate (Sigma-Aldrich) were used for rate-determination studies at four pH values: pH 1.0 (0.2 N HCl/KCl buffer), pH 4.0 (0.1 M Sodium acetate/Acetic Acid buffer), pH 7.0 (0.1 M potassium phosphate buffer) and pH 10.0 (0.1 M Sodium bicarbonate/Sodium hydroxide buffer). The purity of S-nitrosoglutathione was calculated as 94±1.3% by absorbance at 334 nm using the molar extinction coefficient of 900 M⁻¹ cm⁻¹. Assays were performed using a plate reader (Tecan Infinite F500) monitoring absorbance of 2 mM GSNO at 340 nm over a course of 90 minutes in the presence of varying concentrations of sodium phenylsulfinate. Additional experiments confirmed thiosulfonate stability for >5 hours at pH 1, 4, and 7, but hydrolysis at pH 10. GSNO was stable in 6 M urea/PBS for >1 hour. Absorbance data was imported into KaleidaGraph (version 4.02, Synergy Software), and logarithmically fit to the first order exponential decay. The K_(obs) was calculated for each phenylsulfinic acid concentration, and plotted to calculate the rate constant for the overall reaction.

Quantification of products and by-products.

LC-MS analysis was performed using an Agilent Q-TOF mass spectrometer (1-5 ppm accuracy) coupled with the ultra-high pressure HPLC system connected to a photodiode array detector. Compounds and reaction mixtures were injected in 5 μL volumes on an Agilent Zorbax Eclipse plus C18 rapid resolution column (2.1×50 mm, 1.8μ), and separated using the 10-minute gradient starting at 5% and increasing to 100% acetonitrile in 8 minutes. A solution of 500 μM GSNO was made in water (correcting for the 90% purity of GSNO) and allowed to react with various concentrations of 4-methyl-phenylsulfinic acid for 3 hours at ambient temperature before measurement. To generate standard curves, commercial N-hydroxy-4 -methylbenzenesulfonamide (Combi-Blocks) was diluted in LC-MS grade water (pH 6.9) for LC-MS analysis. The thiosulfonate product, of GSNO and 4-methyl-phenylsulfinic acid was purified by semi-preparative HPLC. Extracted ion chromatograms were integrated for quantification of specific ions.

Mammalian Cell Culture.

Human 293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS, JR Scientific) and 1% (v/v) penicillin-streptomycin-glutamine solution (PSQ, Life Technologies). Cells were harvested at 80% confluence in phosphate buffered saline (PBS) (Life Technologies), and lysed by sonication at 4° C. For stable isotope labeling with amino acids in cell culture (SILAC), 293T cells were grown in SILAC DMEM (Thermo), 100 μg/mL [¹³C₆, ¹⁵N₄] L-Arginine-HCl and [¹³C₆,¹⁵N₂] L-Lysine-HCl (Sigma) or L-Arginine-HCl and L-Lysine-HCl (Sigma) for greater than 6 passages.

Gel-based analysis of S-sulfinylation.

Cells and lysates were protected from light and kept on ice throughout the labeling protocol, except when noted. 293T cell pellets were lysed by sonication (4° C., dark, 10% duty cycle, 10 seconds) in 6 M urea/PBS buffer. Lysate protein concentrations were quantified using the Bio-Rad DC assay, and diluted 3 mg/mL in 6 M urea/PBS. Next, lysates were treated with 50 mM iodoacetamide at room temperature for 30 minutes to alkylate all free thiols, and subsequently treated with the specific perturbant (such as ascorbate, hypotaurine etc—as mentioned in the specific experiment). Afterwards, lysates were incubated with 500 μM biotin-hypotaurine or fluorescein-hypotaurine for 30 minutes at room temperature. For in-gel fluorescence analysis, samples were loaded without boiling using non-reducing SDS-PAGE loading buffer for SDS-PAGE separation. Gels were transferred to 0.45 μm polyvinylidine difluoride membrane (Immobilon-P, Millipore) and blocked with 5% bovine serum albumin (BSA, Fisher) in Tris buffered saline-Tween 20 buffer (TBS-T, pH 7.4) for 2 hours at room temperature. After washing, the membrane was incubated and probed with a streptavidin conjugated to DyLight 633 (Thermo, 50 μg/L, 2.5% BSA, 0.02% NaN₃, TBS-T, pH 7.4) for 1 hour at room temperature and washed with TBS-T. For GAPDH detection, blots were probed with the anti-GAPDH mouse monoclonal antibody (mAb 6C5, Calbiochem, 1 μg/mL, 2.5% BSA, 0.02% NaN₃, TBS-T, pH 7.0), washed and probed with a secondary Alexa Fluor 532 nm goal-anti-mouse antibody conjugate (IgG H+L, Life Technologies, 2 μg/mL antibody, 0.06% NaN₃, TBS-T) for 1 hour at room temperature. In-gel fluorescence and streptavidin blots were analyzed using a GE typhoon scanner at appropriate wavelengths (488/526 for fluorescein and 633/670 for Cy5-Streptavidin).

Purification recombinant GAPDH AND DJ-1.

Human GAPDH and DJ-1 cDNAs were amplified from 293T cDNA, cloned into the bacterial 6His expression vector pET45b, and transformed into BL21 E.coli. Bacteria were grown in LB media at 37° C. to an OD600 of 0.6, and induced with 0.4 mM IPTG for 4 additional hours at 37° C. After lysozyme treatment and sonication, the cleared lysate was incubated with Talon resin (Clontech), and loaded on a column. After sufficient washing, the purified recombinant protein was eluted with imidazole and dialyzed into PBS, typically yielding 10-15 mg/L of culture.

LC-MS analysis of purified proteins.

Purified human GAPDH was diluted to 0.2 mg/mL in phosphate buffered saline (pH 7.3), and treated with 20 mM iodoacetamide for 30 minutes. Next, the sample was treated with 100 μM biotin-SO₂H. Purified human DJ-1 was diluted to 0.2 mg/mL in PBS, and incubated with 20 mM iodoacetamide to alkylated free thiols. Next, the sample was treated with 2 mM hydrogen peroxide for 20 minutes to promote Cys106 sulfinic acid formation. The resulting DJ-1 protein was mixed with excess N-acetyl-(S-Nitroso)-Cys-OMe for 30 minutes in the dark at room temperature. After labeling, the separate protein-probe mixtures were dried using a Savant SPD1010 concentrator (Thermo) and reconstituted in 200 μL of 2 M urea in 25 mM ammonium bicarbonate buffer with 2 μL of 100 mM CaCl₂. The reconstituted sampled incubated with mass spectrometry grade Trypsin (Promega, 2 μL of 0.5 mg/mL) for 6 hours at 37° C. with agitation. After trypsin digestion, additional salts were removed using a Waters Oasis HLB glution plates (30 μm) following the manufacturer's protocol. The eluted peptide sample was dried and reconstituted in LC-MS buffer (0.1% formic acid in 3% acetonitrile/water with 5 fmol/μL Saccharomyces cerevisiae alcohol dehydrogenase (P00330)). Tryptic peptides were then separated on the Waters NanoAcquity chromatography system fitted with a 5 μM Symmetry C18 (180 μm×20 mm) trap column and a 1.8 μm High Strength Silica (HSS-T3) analytical column (75 μm×150 mm) over a 80 minute gradient from 5% to 35% acetonitrile, followed by a 85% acetonitrile wash for approximately 10 minutes and re-equilibration to 5% acetonitrile at a flow rate of 0.5 μL/min. Eluted peptides were analyzed using a Waters Synapt G2-S HDMS time-of-flight mass spectrometer in positive mode. Glu-1-Fibrinopeptide B (Glu-Fib) was used as an internal lock-mass. MS data was searched using Waters Protein Lynx Global Server v3.0 using the exact mass calculated for the cysteine thiosulfonate modification.

Labeling and enrichment of cell lysates for LC-MS analysis.

Cells and lysates were protected from light and kept on ice throughout the labeling protocol, except when noted. SILAC paired lysates were prepared separately in 6 M urea/PBS at 3 mg/mL. Approximately 1.2 mg of each lysate was used for each biological replicate. All samples were treated with 50 mM iodoacetamide for 30 minutes to alkylate reduced thiols. Next, “light” and “heavy” lysates were treated with 400 μM of the biotin-SO₂H probe, and the other “light” and “heavy” lysates were treated with 400 μM of the biotin-SO₃H probe. The lysates were allowed to incubate with the probes for 45 minutes in the dark. After 45 minutes, the lysates were quenched by chloroform-methanol addition, and centrifuged to isolate the protein interface while removing excess probe and iodoacetamide. The isolated protein pellet was washed 3 times with cold methanol, each time sonicating the protein pellet and removing the supernatant after centrifugation. The precipitated lysates were re-solubilized in 500 μL of 6 M urea/25 mM ammonium bicarbonate, which required vigorous vortexing and heating (37° C., 10 min). Protein concentrations were measured again using the BioRad DC assay, and normalized to the lowest concentration obtained across the 8 samples. The “light” and “heavy” samples were mixed in a 1:1 ratio (vol/vol), matching a “light” lysate labeled with the biotin-SO₂H probe with a “heavy” lysate labeled with the biotin-SO₃H probe and vice-versa, yielding a total of four independent biological replicates. Each combined sample was transferred to a 15 mL conical tube containing 280 μL of 10% SDS. After brief heating (50° C., 2 min), samples were diluted with 5.5 mL PBS. 100 μL of a 50% streptavidin slurry (Millipore) was washed three times with PBS, and transferred to the solubilized, labeled lysate. Each sample was incubated on a rotary mixer for 2 hours at room temperature, and washed 3 times with 3 mL of 1% SDS in PBS, and 7 times with 3 mL of PBS. The resin was then transferred to a 1.5 mL screw-top conical tube in 200 μL of 2 M urea/25 mM ammonium bicarbonate supplemented with 1 mM aqueous calcium chloride, and 2 μg of trypsin was added to each tube. Samples were incubated 37° C. with agitation for 6-8 hours, and the supernatant was collected and pooled with 2× 150 μL washes, and cleaned up using the Oasis HLB μEglution plates (30 μm). The eluted peptide sample was dried and reconstituted in final 2D-LC-MS buffer, which consists of 20 mM ammonium formate, pH 10.0 buffer with 5 fmol/μL Saccharomyces cerevisiae alcohol dehydrogenase (Waters) as internal standard. Tryptic peptides were stored at −80 ° C. until subjected to LC-MS analysis.

2-D LC-MS of enriched samples.

In-line liquid chromatography of tryptic peptides was performed on the Waters 2D-NanoAcquity chromatography system fitted with a X-BRIDGE BEH130 C18 5 μM (300 μM×50 mm) peptide separation technology fractioning column (column chemistry: 1,2-bis(siloxyethane) [—O₃SiCH₂CH₂SiO₃—]), a 5 μM Symmetry C18 (180 μm×20 mm) trap column and a 1.8 μm High Strength Silica (HSS-T3) analytical column (75 μm×150 mm) using gradients described in Tables 2 and 3. In a 2D chromatography system, tryptic peptides are first loaded onto the fractioning column, followed by fractionation by sequential gradients that gradually increase in the organic component. In the 2D NanoAcquity workflow, peptides are first delivered in a pH 10.0 buffer, and loaded on to the fractioning column (1^(st) dimension) using 20 mM ammonium formate solution as the aqueous phase. The organic phase on the 1^(st) dimension is 100% acetonitrile. In each step, a fraction of the peptides are eluted to the trapping column, and delivered to the analytical column (2^(nd) dimension) for separation. The 2^(nd) dimension uses 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the solvents. During trapping stage, the basic buffers are diluted 1:10 with the acidic solvents to capture the peptide fraction on the trapping column, transferred to the analytical column, and separated over a reverse phase gradient for direct electrospray ionization to the instrument source. Peptides were analyzed using a Waters Synapt G2S HDMS time-of-flight mass spectrometer using ion mobility separation and data independent fragmentation algorithms¹⁷.

TABLE 2 Fractioning Column gradient. Time Flow Rate Step (min) (μL/min) % Water % Acetonitrile 1 Initial 2.0 97 3 2 0.5 2.0 97 3 3 1.0 2.0 89.2 10.8 4 5.0 2.0 89.2 10.8 5 5.5 2.0 97 3 6 20.5 2.0 97 3 Fraction 1 shown. Fractions 2, 3, 4, and 5 reached 14%, 16.7%, 20.4%, and 50% acetonitrile, respectively.

TABLE 3 Analytical Column gradient. Time Flow Rate Step (min) (μL/min) % Water % Acetonitrile 1 Initial 0.5 93 7 2 37.11 0.5 15 85 3 42.11 0.5 15 85 4 43.11 0.5 93 3 5 45.00 0.5 93 7 Common gradient for all 5 fractions in 2D mode. SILAC data analysis.

Raw data files searched against the reviewed human database (Uniprot) using Waters Protein Lynx Global Server (PLGS) version 3.0 at a protein false discovery rate of 4%. Using in-house scripts, all data from technical and biological replicates were merged, removing in-source fragments, unlabeled peptides not containing an arginine or lysine, and any precursors greater than ±5 ppm in mass difference from the calculated theoretical mass. Data was sorted to match SILAC peptides with their corresponding “Heavy” or “Light” partners. Peptides without a pair were labeled as potential “uniques”. Additional data custom scripts were used to calculate the average SILAC ratio, the estimated top3 abundance, the number of SILAC peptides pairs identified for each protein, the SILAC ratio standard deviation, and the number of times the protein was identified across all replicates. The potential “unique” list was further processed to assess their true “uniqueness”. Each unpaired “Light” peptide was assigned a value of “0”; and every unpaired “Heavy” peptide was assigned a value of “1”. If a protein had 10 peptides that were all “light”, then its average “value” from the pivot table analysis is “0”, and assigned as a “unique” protein. Conversely, if all peptides for a protein were “Heavy”, then its average value is “1”. All proteins with a value greater than 0 or less than 1 were deleted from further analysis. If a protein had 50 peptides associated with it, and 40 of those peptides had pairs and 10 did not, then the protein was removed from the “unique” category.

Analysis of unenriched lysates.

293T cells were lysed in 20 mM ammonium bicarbonate containing 2 M urea and the protein concentration was determined. Lysates from two different biological replicates were used diluted to 3 mg/mL, and reduced with 20 mM dithiothreitol (DTT), followed by alkylation with 50 mM iodoacetamide and digested with trypsin. Tryptic digests were injected to a 1D Waters NanoAcquity UPLC system equipped with a 5 μM Symmetry C18 (180 μm×20 mm) trap column and a 1.8 μm High Strength Silica (HSS-T3) analytical column (75 μm×150 mm) using a gradient described in Table 4. Eluted peptides were analyzed using a Waters Synapt G2S HDMS time-of-flight mass spectrometer using ion mobility separation and data-independent fragmentation algorithms¹⁷ (2 biological replicates×5 technical replicates for each biological replicate). Top3 analysis of the identified proteins was performed to compare relative abundance of the identified proteins across different biological and technical replicates.

TABLE 4 Analytical gradient for unenriched proteomics. Time Flow Rate Step (min) (μL/min) % Water % Acetonitrile 1 Initial 0.5 93 7 2 85.00 0.5 65 35 3 90.00 0.5 50 50 4 92.00 0.5 20 80 5 100.00 0.5 20 80 6 102.00 0.5 93 7 7 120 0.5 93 7 Common gradient for 2 biological replicates each with 5 technical replicates. Gene Ontology (GO) analysis for functional annotation.

Gene ontology analysis was performed on both the enriched and the unenriched 293T cell proteomic datasets. Identified proteins were analyzed using the AmiGO term enrichment algorithm, which classified proteins into various GO terms²⁴. The entire Uniprot human database was used as the background dataset and electronically inferred data were excluded from the GO term analysis. The p-value was set at 0.0001 (maximum). Additional validation was performed using the DAVID functional annotation tool (see, e.g., Huang, D. W., Sherman, B. T. & Lempicki, R. A. Nature Protocols 4, 44-57 (2009)).

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the medical sciences are intended to be within the scope of the following claims. 

1-28. (canceled)
 29. A method for detecting protein S-sulfinylation within cysteine residues of a protein, comprising: providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled sulfinic acid moiety, exposing the composition to the biological sample such that an interaction between the labeled sulfinic acid moiety and a S-nitrosothiol side chain of a cysteine residue results in generation of a labeled sulfonothioate moiety, and characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation.
 30. The method of claim 29, further comprising identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation.
 31. The method of claim 29, wherein the labeled sulfinic acid moiety comprises an imaging agent and/or an enrichment tag.
 32. The method of claim 31, wherein said imaging agent is a fluorescent dye.
 33. The method of claim 32, wherein in-gel fluorescence is used to characterize the cysteine residues of a protein having a labeled sulfonothioate moiety as having undergone S-sulfinylation.
 34. The method of claim 31, wherein said enrichment agent is biotin or desthiobiotin.
 35. The method of claim 31, wherein flash chromatography followed by NMR and/or mass spectrometry is used to structurally identify the product of the reaction between a labeled sulfinic acid moiety and a S-nitrosothiol side chain of a cysteine residue.
 36. The method of claim 31, wherein high performance liquid chromatography is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation.
 37. The method of claim 31, wherein mass spectrometry is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation.
 38. The method of claim 29, wherein the one or more proteins having cysteine residues is selected from one or more of the proteins recited in FIG.
 16. 39. A method of identifying pharmaceutical agents capable of inhibiting S-sulfinylation, comprising: providing a pharmaceutical agent and a biological sample comprising one or more proteins having cysteine residues known to undergo S-sulfinylation, exposing the pharmaceutical agent to the biological sample, characterizing the cysteine residues of the one or more proteins as having or not having undergone S-sulfinylation with the method of claim 1, and identifying the pharmaceutical agent as a S-sulfinylation inhibitor if the characterizing indicates an absence of S-sulfinylation.
 40. The method of claim 39, wherein the one or more proteins having cysteine residues known to undergo S-sulfinylation are selected from one or more of the proteins recited in FIG.
 16. 41. A method for detecting protein S-sulfinylation within cysteine residues of a protein, comprising: providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled nitrosothiol moiety, exposing the composition to the biological sample such that an interaction between the labeled nitrosothiol moiety and a sulfinic acid side chain of a cysteine residue results in generation of a labeled sulfonothioate moiety, and characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation.
 42. The method of claim 41, further comprising identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation.
 43. The method of claim 41, wherein the labeled nitrosothiol moiety comprises an imaging agent and/or an enrichment tag.
 44. The method of claim 43, wherein said imaging agent is a fluorescent dye.
 45. The method of claim 44, wherein in-gel fluorescence is used to characterize the cysteine residues of a protein having a labeled sulfonothioate moiety as having undergone S-sulfinylation.
 46. The method of claim 43, wherein said enrichment agent is biotin or desthiobiotin.
 47. The method of claim 43, wherein flash chromatography followed by NMR and/or mass spectrometry is used to structurally identify the product of the reaction between a labeled nitrosothiol moiety and a sulfinic acid side chain of a cysteine residue.
 48. The method of claim 43, wherein high performance liquid chromatography is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation.
 49. The method of claim 43, wherein mass spectrometry is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation. 